Embryonic stem cells and the molecular methods to induce their formation from somatic cells has been the subject of intense study in recent years. These techniques allow scientists to bypass the ethically contentious issue of using human embryos and create personalized pluripotent stem cells. These advances underlie the incredible push to begin using these cells for therapeutic purposes. Unfortunately, our ability to generate cells or tissues for therapeutic use is still quite limited. The Blelloch lab has recently shown tht miRNAs are necessary for differentiation of embryonic stem cells, and that specific classes of miRNAs function through opposing pathways to mediate differentiation out of the pluripotent state. This finding suggests that miRNAs are important global regulators of differentiation. In thi application, I hypothesize that the mir302 cluster of miRNAs is critical in the transition from pluripotency to differentiated germ layers by regulating transcripts that mechanistically underlie this critically important cell-fate decision. To test this hypothesis I will first determine the invivo role of mir302 miRNAs by characterizing epiblast formation and gastrulation during embryonic development of mir302 deficient mice. I will then characterize differentiation defects in mouse embryonic stem cells lacking mir302. Next, I will identify and functionally test the molecular targets of mir302 to identify the mechanism through which mir302 regulates germ layer specification. Finally, I will dissect the function of miRNAs containing different seed sequences within the mir302 locus to understand how co- expressed miRNAs with different targets function in development. These experiments will provide detailed information on how miRNAs regulate cell fate decisions and through what mechanism a cell transitions from pluripotency to the primary embryonic germ layers. PUBLIC HEALTH RELEVANCE: Statement Our ability to use embryonic stem cells for regenerative medicine is limited by our understanding of how to generate therapeutically useful cells and tissues. Better understanding of how a unique class of molecules termed micro-RNAs is involved in this process will lead to better therapies that treat or cure human disease and restore lost function.